Inner and outer dynein arms are lost in Johnston's organ sensory cilia of tilB mutants. Cross-sectional electron micrographs are shown of Johnston's organ scolopidia of a y w control fly (A) and a tilB² mutant (B) at the level between the basal body and the ciliary dilation, the ciliary segment that normally contains dynein arms. Enlarged views of the boxed regions of A and B are shown in A′ and B′. Outer dynein arms (arrow) and inner dynein arms (arrowhead) are present in the control but absent in tilB² flies. Bar: 500 nm.

tilB corresponds to CG14620 and encodes a leucine-rich repeat protein. (A) Molecular map of the 1.3-Mbp region including tilB. tilB/CG14620 is located in a gene-rich cluster at the base of the X chromosome, flanked by transposon-rich intervals (transposons not indicated). Shaded bars, predicted gene spans; solid bars, molecularly and genetically identified loci. (B) Deficiency complementation map of the 19E–20B interval. Bars indicate a failure to complement. Complementation data for tilB are from this work; data for other loci are from FlyBase. The position of tilB relative to the molecularly undefined loci wap and intro is still unresolved, reflecting an inconsistency between the complementation patterns of Df(1)DCB1-35c, which deletes or disrupts flam, wap, and intro but not tilB, and Df(1)R38, which deletes wap, intro, and more proximal loci. In addition to the aberrations shown, tilB also is disrupted or deleted in Df(1)GA22, Df(1)48-2, and Df(1)R35; is duplicated in Dp(1;Y)67g19.1; and complements Df(1)16-3-22, Df(1)S54, Df(1)17-25, and Df(1)JC77. (C) The annotated gene CG14620, which lies between CG14614 and CG14615, was identified as tilB. The locations of sequence changes corresponding to the two alleles, tilB¹ and tilB², are shown as single and double asterisks, respectively. The genomic fragment used for transposon-mediated rescue, the upstream regulatory region used for generating tilB-Gal4, and the fragment used for testing expression by RT–PCR are indicated. (D) tilB encodes a 395-amino-acid protein. tilB¹ changes Q171 to a stop codon and tilB² is an in-frame deletion removing an alanine and a tyrosine at position 313–314. The protein has three conserved domains including a leucine-rich repeat (LRR) region in the N-terminal end (residues 16–145), a central coiled-coil domain (residues 171–191), and a region that we refer to as the TilB domain (residues 202–334). (E) Rescue of hearing in tilB mutants with a P-element transgene containing the CG14620 genomic fragment, indicated in C. Representative traces are shown of sound-evoked potentials (SEPs) recorded from the antennal nerves of flies of the indicated genotypes, where P indicates the rescuing transgene inserted in the third chromosome. (F) Quantitative rescue of hearing in both tilB mutants. Flies with the P{CG14620} third chromosome transgene (indicated by P) have SEP amplitudes not significantly different from those of heterozygote females (indicated by +). N = 15 for each genotype. Error bars indicate SEM.

tilB is expressed in ciliated cells. (A) RT–PCR of tilB, using primers for the fragment indicated in , shows expression in the heads and testes. The expected genomic and spliced sizes are indicated. A band of the expected size is amplified from genomic DNA (gDNA). For each tissue, amplification is shown from cDNA using reverse transcriptase (RT) and from the control with this enzyme omitted (−). (B and C) Late stage embryos carrying the tilB-Gal4 driver and UAS-GFP show tilB expression in the lateral pentascolopidial organs (arrows in B). Salivary gland expression (arrowhead in B) is commonly seen in Gal4 insertion lines (; ) and therefore this expression is most likely spurious. (D) Fluorescent view of adult antennal regions shows that tilB-Gal4 also drives UAS-GFP expression in Johnston's organ (white arrows), which is housed within the second antennal segments (2). The third antennal segments are also indicated (3). (E) tilB-Gal4-driven UAS-TilB:EGFP shows expression in the olfactory sensillae of the third antennal segment. Bars: B and D, 100 μm; C and E, 50 μm.

TilB fusion proteins rescue hearing and are present in chordotonal neuronal cell bodies and dendrites. (A) Two full-length and four truncated fusion proteins were constructed that contain the leucine-rich repeat region (LRR) alone, the LRR and coiled-coil domain (CC), the CC together with the TilB domain (CCTilBD), or the TilB domain alone (TilBD). Those polypeptides that retained the N-terminal LRR region were tagged with Myc (M), while those that retained the C-terminal TilB domain were tagged with GFP (G). (B) Electrophysiology shows that the full-length M:TilB and TilB:G fusions rescue hearing in tilB¹ and tilB² mutants (red box). The full-length fusion proteins rescue without the tilB-Gal4 driver, indicating that a low level of protein allowed by the UAS sequences is sufficient for rescue. Each bar in the graph depicts the mean + SEM, with N = 15. (C–F) Expression of GFP fusion proteins in embryonic lch5 organs with the tilB-Gal4 driver. (C) The full-length TilB:G fusion protein is present throughout the cytoplasm including the inner and outer dendritic segments. (D) The CC-TilBD:G fusion is similarly distributed, as are the TilBD:G (E) and free GFP alone (F). (G–J) Johnston's organ expression of the same constructs as in C–F with the elav-Gal4 driver, counterstained with phalloidin (red). Arrowheads indicate the basal body region at the junction of the inner and outer dendritic segments, while arrows point to the ciliary dilations. Bar in C: 10 μm for C–F. Bars in G–J: 25 μm.

Expression of TilB with a germline-compatible vector. (A) An N-terminal Venus (EYFP)-tagged TilB fusion protein generated in the pUASP vector rescues deafness only in the presence of a Gal4 driver. Flies with the driver but without V:TilB are deaf, as are flies with V:TilB but without the driver. This is in contrast to TilB:G, which rescues deafness even in the absence of the driver. (B and C) To ensure that overexpression of full-length TilB fusion proteins did not saturate the cell, the full-length fusion proteins were expressed under the control of the tilB-Gal4 driver. The results still show that the protein is present in the inner dendritic segment and basal body (arrowheads). V:TilB and TilB:G were also present in the outer dendritic segment (arrows). Bars: 20 μm. (D) Transgenic flies with V:TilB created using the pUASP vector show TilB localization to the sperm flagella when driven by tilB-Gal4. (E) V:TilB showing presence in testes. Bar: 200 μm. E shows V:TilB in sperm bundle in testes and F shows V:TilB in sperm released from testes. Bars in E and F: 50 μm.

tilB is conserved in ciliated eukaryotes and co-occurs with axonemal dyneins. This figure is based on Figure 4 of ; data in black are from that work. Columns indicate proteins representing axonemal dynein subunits, including dynein heavy chains (DHC) and intermediate chains (IC) from the outer (ODA) and inner (IDA) dynein arms, and from the intraflagellar transport (IFT) pathway, including the IFT dynein (DYNCH2). Rows list representative unikont and bikont eukaryotes with sequenced genomes. Each solid circle indicates a tilB ortholog in that species, as determined by the top score in reciprocal BLASTP or TBLASTN searches. Open circle: no homolog present. TilB orthologs, accession numbers, and E-values are as follows: Homo, Homo sapiens testis-specific leucine-rich repeat protein (LRRC6; AAB02976), 8e-80; Takifugu, Takifugu rubripes (Ensembl release 56) scaffold 3605, 4e-40 (partial); Chlamydomonas, Chlamydomonas reinhardtii protein ID 107408, 8e-37 (partial); Ostreococcus, Ostreococcus lucimarinus ABO94938.1, 3e-53 and O. tauri CAL52896.1, 1e-52; Plasmodium, Plasmodium knowlesii, CAQ42230.1, 4e-50; Toxoplasma, Toxoplasma gondii EEA99420.1, 2e-44; Tetrahymena, Tetrahymena thermophila EAR92110.1, 4e-60; Thalassiosira, Thalassiosira pseudonana EED89058.1, 3e-45; Phythopthora, Phythopthora sojae scaffold 12 90634-90933, 1e-32; Trypanosoma, Trypanosoma brucei AAX80642.1, 9e-55; Giardia, Giardia intestinalis EET00698, 3e-43.